Radiation therapy, the therapeutic use of ionizing radiation to induce damage to the DNA, plays an important role in cancer management. DNA lesions are recognized by cell cycle checkpoints, leading to activation of DNA damage repair pathways. Recently, cancer stem cells have been shown to promote tumor radioresistance through activation of DNA damage response. In addition, a trans-differentiation process, termed epithelial-mesenchymal transition (EMT), is thought to promote metastasis and generate stem-like cells. Besides its implication in tumor progression and metastasis, EMT has been shown to be associated with characteristics of cancer stem cells, including chemoresistance and radioresistance. However, it is not clear which EMT regulators play causal roles in these properties. We and others have previously uncovered microRNA-mediated regulation of metastasis and EMT. Moreover, we provided proof-of-principle evidence that therapeutic silencing of a pro-metastatic microRNA can block metastasis in a preclinical model. In this research, we intend to seek EMT-regulating transcription factors and microRNAs that represent novel regulators of radioresistance and DNA damage repair, determine their mechanism of action and regulation of expression, and explore their potential use as new cancer biomarkers and therapeutic targets. In preliminary studies, we found that two recently reported EMT regulators, ZEB1 and miR-205, negatively regulate each other and play opposing roles in modulating radiosensitivity of tumor cells. Furthermore, our data point to a role of ATM signaling in stabilizing ZEB1 and a role of ZEB1 in promoting deubiquitination-mediated stabilization of CHK1, a protein kinase that is required for cell cycle checkpoint control and homologous recombination-mediated DNA damage repair. In proposed future studies, we will: 1) investigate the role of ZEB1 and miR-205 in tumor radioresistance and cancer stem cell properties; 2) identify the mechanism by which ZEB1 regulates radiosensitivity and DNA damage response; 3) determine how ZEB1 is upregulated in response to radiation and DNA damage; 4) study the involvement of ZEB1 and miR-205 in human tumors and the therapeutic potential. The knowledge gained from these studies will provide new insights into how specific EMT regulators contribute to tumor radioresistance and DNA damage repair and may have significant clinical implications.